Plant Somatic Embryogenesis
Somatic embryogenesis in plants is a process in which somatic embryos are formed from somatic cells of plant tissue, usually an explant in vitro. The somatic embryos formed are genetically identical copies of the plant providing the initial explant. The process of somatic embryogenesis thereby offers a tool to obtain large numbers of genotypically identical plants for multiplication of selected genotypes of commercial interest, for conservation of endangered species or for generating genetically uniform plant material for research purposes.
Somatic embryogenesis is of special interest with regard to coniferous trees. Conifers of economic importance have long generation times, so that breeding through crossing and selection proceeds very slowly. Conifers are wind-pollinated over long distances and are highly heterozygous, so that the offspring of selected individuals is highly variable. Self-pollination, leading to greater homozygosity and uniformity of the offspring, is possible in principle but undesirable in practice owing to pronounced inbreeding depression. Multiplication of selected or elite individuals by somatic embryogenesis offers an attractive solution to these problems. For some species, particularly spruces, the procedure works well on a laboratory scale, but at present sexual propagation via seeds is the only cost-effective method for propagation. This situation could in principle be changed by simplifying and automating the laboratory procedures but several major challenges remain.
Methods for Somatic Embryogenesis
Plant regeneration through somatic embryogenesis in e.g. conifers consists of a series of consecutive steps (see e.g. von Arnold S, Clapham D. Spruce embryogenesis. 2008. Methods Mol Biol. 2008; 427:31-47;, Belmonte M F, Donald G, Reid D M, Yeung E C and Stasolla C. 2005. Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca). J Exp Bot,, Vol. 56,, No. 419,, pp. 2355-2364).
Taking Norway spruce as an example, the step 1, is initiation of an embryogenic culture from an explant, usually a mature or immature embryo, though there are reports of successful initiation from buds from adult trees. This step is carried out on a plant culture medium containing plant growth regulators, usually an auxin and a cytokinin. For step 2, continued proliferation, the initiated cultures are usually subcultured on essentially the same medium as for initiation. At this stage, the proliferating cell masses consist of more or less well differentiated immature somatic embryos, which morphologically correspond to a stage found in the developing seed in the early phase of seed development. The somatic embryos do not undergo any further development during proliferation and mature embryos are not formed during this phase.
In order to obtain embryo maturation, embryogenic cultures are usually transferred to a culture medium where auxin and cytokinin are omitted for step 3, known as prematuration. After about three to seven days of prematuration, during which the influences of auxin and cytokinin are lost, the prematured cultures are transferred to a maturation medium containing abscisic acid (ABA) for step 4, maturation. One characteristic of prematuration stage embryos is that the somatic embryos at this stage lack root meristems.
Step 5 involves partial drying of the embryos under high humidity, which is thought to partly mimic the natural drying of zygotic embryos in the seed and to improve subsequent conversion to plantlets. On a laboratory scale the embryos are picked out by hand using forceps and transferred to a small sterile petri dish. This is then placed in a larger petri dish containing sterile water to raise the humidity. After this step, the embryos are germinated in culture medium and start growing roots. In the final step 6, the embryos are converted into plantlets, separately or in combination with transfer to non-sterile cultivation in pots or containers in a greenhouse.
These six steps are briefly summarized in Table 1, below.
Growth media(importantSomatic embryoStepcomponent/s)characteristics1InitiationPlant growthProembryonic massesregulatorsdevelop from embryoexplants2ProliferationThe same mediaProembryonic massesas step 1proliferate as ‘callus’3PrematurationAll plant growthTransition from(~1 week)regulatorsproembryo to embryo; noare removed.organized shoot and rootmeristems4EarlyInitiated withEmbryos enlarge; shootmaturationabscisic acidand root meristems(3-5 weeks)organize; cotyledons justvisible in some embryosat end of step.5Late maturationContinue withFurther embryoabscisic acid;enlargement, andsuitable stage fordevlopment of meristemsenhanced sugarand vascular tissue;alcohol treatmentcotyledons expand andaccording toseparate.the presentinvention.6Germination toMedium lacksRoot growth, extension ofplantletsgrowthhypocotyl andregulatorscotyledons, greening.
With regard to the above discussion of prior art, the invention addresses in particular the following issues with present somatic embryo maturation methods:    (1) The variable quality of the mature somatic embryos. A considerable fraction, perhaps 50%, of the embryos exposed to maturation medium, is incapable of conversion to rooted plantlets. The most promising embryos can be selected visually, so that around 90% of the selected embryos can be converted to rooted plantlets growing on sterile medium in petri dishes. Of these, some grow slowly so that at planting-out time, only about 75% of the selected embryos are suitable for transfer to greenhouse conditions. Thus there is a need for methods providing improved quality of somatic embryos, in particular in terms of uniformity of the quality.    (2) The unsynchronized development of the embryos exposed to maturation medium. The fraction of embryos that are suitable for conversion do not normally develop synchronously. Furthermore, by the time the slowly developing embryos have reached maturity, most of the fast developers have proceeded to premature germination, which impairs the quality of subsequent conversion to plantlets. It is necessary either to sacrifice the late developers, or harvest the embryos on two or more separate occasions. Thus there is a need for methods achieving synchronized maturation of somatic embryos and need for methods that prevent premature germination.    (3) Partial drying of the mature somatic embryos under high humidity. This step may be problematic, it adds to the labor involved and it would be advantageous if it could be omitted. Thus, there is need for streamlined methods that can dispense with the partial drying step.
It is therefore an object of the invention to provide methods to address the above-identified issues.
It is important to keep in mind that if industrial-scale production of conifers via somatic embryogenesis was used to produce a major fraction of the conifers planted yearly, the production scale would be in the hundreds of millions of plantlets annually. Thus, even relatively small improvements to the process may have significant economic impact.
Definitions
The terms somatic embryo and somatic plant embryo are used interchangeably. The terms refer to plant embryos derived from somatic tissue of a plant.
Norway spruce is a spruce species with the Latin name Picea abies, native to Europe.
The term sugar alcohol refers to a hydrogenated form of a sugar carbohydrate, whose carbonyl group (aldehyde or ketone of a reducing sugar) has been reduced to a primary or secondary hydroxyl group. Examples of sugar alcohols include: Glycol, Glycerol, Erythritol, Threitol. Arabitol, Xylitol, Ribitol, Mannitol, Sorbitol, Dulcitol, Iditol, inositol, Isomalt, Maltitol, Lactitol, Polyglycitol.
Inositol or cyclohexane-1,2,3,4,5,6-hexol is a sugar alcohol having the formula C6H12O6, or (—CHOH—)6. It has nine possible stereoisomers including cis-1,2,3,5-trans-4,6-cyclohexanehexol, or myo-inositol.

Plasmolysis is the process in plant cells where the plasma membrane pulls away from the cell wall due to the loss of water through osmosis, when the cell is exposed to hypertonic medium.
Plasmolytic concentration of a compound is the smallest concentration of said compound in a culture media that is sufficient to induce plasmolysis in plant cells cultured in said culture media.
The related term “near plasmolytic concentration” relates to a concentration that is somewhat below or above the plasmolytic concentration. Preferably, the near plasmolytic concentration is 50-150% of the plasmolytic concentration. More preferably the near plasmolytic concentration is 80-120% of the plasmolytic concentration, even more preferably 90-110% or higher, most preferably at 95-105%.